Time-resolved fluorescence polarization measurements

Dr can be determined by time-resolved fluorescence polarization measurements, either by pulse fluorometry from the recorded decays of the polarized components I l and 11, or by phase fluorometry from the variations in the phase shift between J and I as a function of frequency (see Chapter 6). If the excited-state lifetime is unique and determined separately, steady-state anisotropy measurements allow us to determine Dr from the following equation, which results from Eqs (5.10) and (5.41) ... 

This relation shows that the rotational correlation time is uncoupled from the excited-state lifetime, in contrast to classical steady-state or time-resolved fluorescence polarization measurements (see Chapter 5). The important consequence is the possibility of observing slow rotations with fluorophores of short lifetime. This is the case for biological macromolecules labeled with fluorophores (e.g. rhodamine) whose lifetime is of a few nanoseconds. 

Time-resolved fluorescence polarization measurements for 1 and 2 in n-heptane showed that after pulsed excitation, the initial value of the fluorescence anisotropy is about 0.25. This... 

The changes have been used to provide information about the enviromnent of the fluorescent probe and to follow changes in conformation of the macromolecule. In other work the study of the fluorescence polarization properties of the attached probe under steady state illumination and the application of Perrin s equation enable calcu-latnn of the rotary Brownian motion of the polymer. This technique has been extended by Jablonski and Wahl to the use of time-resolved fluorescence polarization measurements to calculate rotational relaxation times of molecules These experiments are discussed fiilly in the fdlowing section of this review. 

Fluorescence Polarization Studies of PMA and PAA. Time-resolved fluorescence polarization measurements are potentially a powerful means for studying molecular mobility. The fluorescence anisotropy function r(t) may be generated by monitoring the decay of vertically (Iv(t)) and horizontally (Ijj(t)) polarized components of emission following excitation by vertically polarized light pulses (Equation 1). 

L.A. Kelly, J.G. Trunk, J.C. Sutherland, Time-resolved fluorescence polarization measurements for entire emission spectra with a resisitive-anode, single-photon-counting detector The fluoreseence omnilizer, Rev. Sci. Instrum. 68, 2279-2286 (1997)... 

An Applied Photophysics Model SP 2X nanosecond spectrometer incorporating an alternating polarization rotation unit ( ) was used for the time-resolved fluorescence anisotropy measurements. An excitation wavelength of 365 nm was employed for excitation of the anthracene end-groups and emission above 400 nm was isolated with a Schott GG 400 filter. 

A synchronously pumped DCM dye laser was used as the picosecond light source in the pump-probe experiments. A Soleil-Babinet compensator in the excitation beam was set so that the polarization of the excitation and probe beams were parallel or perpendicular. Fluorescence anisotropies were measured with a Spex fluorolog spectrometer. The time resolved fluorescence was measured by single photon counting. 

The simplest fluorescence measurement is that of intensity of emission, and most on-line detectors are restricted to this capability. Fluorescence, however, has been used to measure a number of molecular properties. Shifts in the fluorescence spectrum may indicate changes in the hydrophobicity of the fluorophore environment. The lifetime of a fluorescent state is often related to the mobility of the fluorophore. If a polarized light source is used, the emitted light may retain some degree of polarization. If the molecular rotation is far faster than the lifetime of the excited state, all polarization will be lost. If rotation is slow, however, some polarization may be retained. The polarization can be related to the rate of macromolecular tumbling, which, in turn, is related to the molecular size. Time-resolved and polarized fluorescence detectors require special excitation systems and highly sensitive detection systems and have not been commonly adapted for on-line use. 

Fig. 22 (a) Comparison of fluorescence lifetime (blue triangles), calculated from (13), and measured by time-resolved fluorescence red circles) as a function of solvent polarity for G19. (b) Fluorescence quantum yield blue squares) and peak ground state absorption wavelength red circles) as a function of solvent polarity given by the percentage of toluene (T) in toluene-ACN mixtures for G19... 

Situation with H-bonding also demands to take into account the fact that alcohols have ability to form various associates or even clusters at normal conditions. The most efficient method for determination of inhomogeneity in the excited states is fluorescence polarization measurements. These methods also frequently applied for studying of solvent viscosity, they may be provided in two variants steady state and time-resolved. Relations for time-resolved and steady state fluorescence anisotropy may be given as [1, 2, 75] ... 

In addition to fluorescence intensity and polarization, fluorescence spectroscopy also includes measurement of the lifetime of the excited state. Recent improvements in the design of fluorescence instrumentation for measuring fluorescence lifetime have permitted additional applications of fluorescence techniques to immunoassays. Fluorescence lifetime measurement can be performed by either phase-resolved or time-resolved fluorescence spectroscopy. 

The optical setup for time-resolved micro-luminescence measurements is based around an Axiotech 100 HD Zeiss microscope, modified to allow laser injection and fluorescence collection. The sample is observed either under transmission or reflection of polarized white light, or under UV illumination (HBO lamp). A set-up consisting of a dichroic mirror, for the selection of the excitation wavelength, and an objective (Epiplan Neofiuar obj. > 350 nm Ealing/Coherent reflection obj. < 350 nm) is used to focus the laser beam on the sample (spatial resolution over 5 pm with a x50 objective). The lim-... 

Experimentally, these effects are tested by fluorescence and absorption measurements. These directly probe solvent polarization dynamics on molecular time-scales [100, 101]. For instance, the time resolved fluorescence spectrum of a chromophore, whose excited state dipole moment is subject to interactions with the surrounding solvent molecules, will exhibit fluorescence spectra that are strongly solvent dependent. The solvent molecules attempt to compensate the changes of charge density in the chromophore and, in sum, the fluorescence... 

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